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DOE OSTI2019

Crystal engineering with DNA

This Review chronicles over two decades of research into creating a genetic code for crystal engineering. Rather than directing biological processes, this code uses synthetic forms of DNA to programme the assembly of nanoparticles and microparticles into 1D, 2D and 3D crystalline architectures, in which almost every aspect of the resultant structures can be systematically controlled. Within this conceptual framework, the structural and functional advances are described in an effort to define the present level of sophistication and to predict future directions of the platform. These advances include exotic structures with programmable lattice symmetries and well-defined crystal habits, responsive materials that leverage the intrinsic properties of nucleic acids to manipulate structures on demand, nanoparticle superlattices grown epitaxially from surfaces and colloidal crystals that offer insights into light–matter interactions. Looking forward, we challenge the community to leverage the extraordinary structural control afforded by crystal engineering with DNA to synthesize classes of functional materials that push beyond what has been possible with naturally occurring crystalline materials or those made by more conventional strategies.

catalysis (homogeneous), solar (photovoltaic), bio↗

DOE OSTI2019

Patterning non-equilibrium morphologies in stimuli-responsive gels through topographical confinement

Stimuli-responsive “smart” polymers have generated significant interest for introducing dynamic control into the properties of antifouling coatings, smart membranes, switchable adhesives and cell manipulation substrates. Switchable surface morphologies formed by confining stimuli-responsive gels to topographically structured substrates have shown potential for a variety of interfacial applications. Beyond patterning the equilibrium swelling behavior of gels, subjecting stimuli-responsive gels to topographical confinement could also introduce spatial gradients in the various timescales associated with gel deformation, giving rise to novel non-equilibrium morphologies. In this work, we show how by curing poly(N-isopropylacrylamide) (pNIPAAm)-based gel under confinement to a rigid, bumpy substrate, we can not only induce the surface curvature to invert with temperature, but also program the transient, non-equilibrium morphologies that emerge during the inversion process through changing the heating path. Finite element simulations show that the emergence of these transient morphologies is correlated with confinement-induced gradients in polymer concentration and position-dependent hydrostatic pressure within the gel. To illustrate the relevance of such morphologies in interfacial applications, we show how they enable us to control the gravity-induced assembly of colloidal particles and microalgae. Finally, we show how more complex arrangements in particle assembly can be created through controlling the thickness of the temperature-responsive gel over the bumps. Patterning stimuli-responsive gels on topographically-structured surfaces not only enables switching between two invertible topographies, but could also create opportunities for stimuli ramp-dependent control over the local curvature of the surface and emergence of unique transient morphologies. Harnessing these features could have potential in the design of multifunctional, actuatable materials for switchable adhesion, antifouling, cell manipulation, and liquid and particle transport surfaces.

42 ENGINEERING↗

DOE OSTI2017

H/D exchange mass spectrometry and statistical coupling analysis reveal a role for allostery in a ferredoxin-dependent bifurcating transhydrogenase catalytic cycle

Recent investigations into ferredoxin-dependent transhydrogenases, a class of enzymes responsible for electron transport, have highlighted the biological importance of flavin-based electron bifurcation (FBEB). FBEB generates biomolecules with very low reduction potential by coupling the oxidation of an electron donor with intermediate potential to the reduction of high and low potential molecules. Bifurcating systems can generate biomolecules with very low reduction potentials, such as reduced ferredoxin (Fd), from species such as NADPH. Metabolic systems that use bifurcation are more efficient and confer a competitive advantage for the organisms that harbor them. Structural models are now available for two NADH-dependent ferredoxin-NADP + oxidoreductase (Nfn) complexes. These models, together with spectroscopic studies, have provided considerable insight into the catalytic process of FBEB. However, much about the mechanism and regulation of these multi-subunit proteins remains unclear. Using hydrogen/deuterium exchange mass spectrometry (HDX-MS) and statistical coupling analysis (SCA), we identified in this work specific pathways of communication within the model FBEB system, Nfn from Pyrococus furiosus , under conditions at each step of the catalytic cycle. HDX-MS revealed evidence for allosteric coupling across protein subunits upon nucleotide and ferredoxin binding. SCA uncovered a network of co-evolving residues that can provide connectivity across the complex. Together, the HDX-MS and SCA data show that protein allostery occurs across the ensemble of iron-sulfur cofactors and ligand binding sites using specific pathways that connect domains allowing them to function as dynamically coordinated units.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2017

Subcellular pigment distribution is altered under far-red light acclimation in cyanobacteria that contain chlorophyll $\mathcal{f}$

Far-Red Light (FRL) acclimation is a process that has been observed in cyanobacteria and algae that can grow solely on light above 700 nm. The acclimation to FRL results in rearrangement and synthesis of new pigments and pigment-protein complexes. Here, cyanobacteria containing chlorophyll $\mathcal{f}$, Synechococcus sp. PCC 7335 and Halomicronema hongdechloris , were imaged as live cells with confocal microscopy. H. hongdechloris was further studied with hyperspectral confocal fluorescence microscopy (HCFM) and freeze-substituted thin-section transmission electron microscopy (TEM). Under FRL, phycocyanin-containing complexes and chlorophyll-containing complexes were determined to be physically separated and the synthesis of red-form phycobilisome and Chl $\mathcal{f}$ was increased. The timing of these responses was observed. The heterogeneity and eco-physiological response of the cells was noted. Additionally, a gliding motility for H. hongdechloris is reported.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2015

A Versatile Click-Compatible Monolignol Probe to Study Lignin Deposition in Plant Cell Walls

Here, lignin plays important structural and functional roles in plants by forming a hydrophobic matrix in secondary cell walls that enhances mechanical strength and resists microbial decay. While the importance of the lignin matrix is well documented and the biosynthetic pathways for monolignols are known, the process by which lignin precursors or monolignols are transported and polymerized to form this matrix remains a subject of considerable debate. In this study, we have synthesized and tested an analog of coniferyl alcohol that has been modified to contain an ethynyl group at the C-3 position. This modification enables fluorescent tagging and imaging of this molecule after its incorporation into plant tissue by click chemistry-assisted covalent labeling with a fluorescent azide dye, and confers a distinct Raman signature that could be used for Raman imaging. We found that this monolignol analog is incorporated into in vitro -polymerized dehydrogenation polymer (DHP) lignin and into root epidermal cell walls of 4-day-old Arabidopsis seedlings. Incorporation of the analog in stem sections of 6-week-old Arabidopsis thaliana plants and labeling with an Alexa-594 azide dye revealed the precise locations of new lignin polymerization. Results from this study indicate that this molecule can provide high-resolution localization of lignification during plant cell wall maturation and lignin matrix assembly.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2017

Determination of Cell Doubling Times from the Return-on-Investment Time of Photosynthetic Vesicles Based on Atomic Detail Structural Models

Cell doubling times of the purple bacterium Rhodobacter sphaeroides during photosynthetic growth are determined experimentally and computationally as a function of illumination. For this purpose, energy conversion processes in an intracytoplasmic membrane vesicle, the chromatophore, are described based on an atomic detail structural model. The cell doubling time and its illumination dependence are computed in terms of the return-on-investment (ROI) time of the chromatophore, determined computationally from the ATP production rate, and the mass ratio of chromatophores in the cell, determined experimentally from whole cell absorbance spectra. The ROI time is defined as the time it takes to produce enough ATP to pay for the construction of another chromatophore. The ROI time of the low light-growth chromatophore is 4.5–2.6 h for a typical illumination range of 10–100 μmol photons m –2 s –1 , respectively, with corresponding cell doubling times of 8.2–3.9 h. Finally, when energy expenditure is considered as a currency, the benefit-to-cost ratio computed for the chromatophore as an energy harvesting device is 2–8 times greater than for photovoltaic and fossil fuel-based energy solutions and the corresponding ROI times are approximately 3–4 orders of magnitude shorter for the chromatophore than for synthetic systems.

09 BIOMASS FUELS↗

DOE OSTI2016

Quinone 1 e – and 2 e – /2 H + Reduction Potentials: Identification and Analysis of Deviations from Systematic Scaling Relationships

Quinones participate in diverse electron transfer and proton-coupled electron transfer processes in chemistry and biology. To understand the relationship between these redox processes, an experimental study was carried out to probe the 1 e – and 2 e – /2 H + reduction potentials of a number of common quinones. The results reveal a non-linear correlation between the 1 e – and 2 e – /2 H + reduction potentials. This unexpected observation prompted a computational study of 134 different quinones, probing their 1 e – reduction potentials, p K a values, and 2 e – /2 H + reduction potentials. We report the density functional theory calculations reveal an approximately linear correlation between these three properties and an effective Hammett constant associated with the quinone substituent(s). However, deviations from this linear scaling relationship are evident for quinones that feature intramolecular hydrogen bonding, halogen substituents, charged substituents, and/or sterically bulky substituents. These results, particularly the different substituent effects on the 1 e – versus 2 e – /2 H + reduction potentials, have important implications for designing quinones with tailored redox properties.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2016

Fast Photochemical Oxidation of Proteins Maps the Topology of Intrinsic Membrane Proteins: Light-Harvesting Complex 2 in a Nanodisc

Although membrane proteins are crucial participants in photosynthesis and other biological processes, many lack high-resolution structures. Prior to achieving a high-resolution structure, we are investigating whether MS-based footprinting can provide coarse-grained protein structure by following structural changes that occur upon ligand binding, pH change, and membrane binding. Our platform probes topology and conformation of membrane proteins by combining MS-based footprinting, specifically fast photochemical oxidation of proteins (FPOP), and lipid Nanodiscs, which are more similar to the native membrane environment than are the widely used detergent micelles. We describe here results that show a protein’s outer membrane regions are more heavily footprinted by OH radicals whereas the regions spanning the lipid bilayer remain inert to the labeling. Nanodiscs generally exhibit more protection of membrane proteins compared to detergent micelles and less shielding to those protein residues that exist outside the membrane. The combination of immobilizing the protein in Nanodiscs and footprinting with FPOP is a feasible approach to map extra-membrane protein surfaces, even at the amino-acid level, and to illuminate intrinsic membrane protein topology.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2020

Cellulose synthase interactive1- and microtubule-dependent cell wall architecture is required for acid growth in Arabidopsis hypocotyls

Auxin-induced cell elongation relies in part on the acidification of the cell wall, a process known as acid growth that presumably triggers expansin-mediated wall loosening via altered interactions between cellulose microfibrils. Cellulose microfibrils are a major determinant for anisotropic growth and they provide the scaffold for cell wall assembly. Little is known about how acid growth depends on cell wall architecture. To explore the relationship between acid growth-mediated cell elongation and plant cell wall architecture, two mutants (jia1-1 and csi1-3) that are defective in cellulose biosynthesis and cellulose microfibril organization were analyzed. The study revealed that cell elongation is dependent on CSI1-mediated cell wall architecture but not on the overall crystalline cellulose content. We observed a correlation between loss of crossed-polylamellate walls and loss of auxin- and fusicoccin-induced cell growth in csi1-3. Furthermore, induced loss of crossed-polylamellate walls via disruption of cortical microtubules mimics the effect of csi1 in acid growth. We hypothesize that CSI1- and microtubule-dependent crossed-polylamellate walls are required for acid growth in Arabidopsis hypocotyls.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2016

Investigating Biochemical and Developmental Dependencies of Lignification with a Click-Compatible Monolignol Analog in Arabidopsis thaliana Stems

Lignin is a key structural component of plant cell walls that provides rigidity, strength, and resistance against microbial attacks. This hydrophobic polymer also serves a crucial role in water transport. Despite its abundance and essential functions, several aspects of lignin biosynthesis and deposition remain cryptic. Lignin precursors are known to be synthesized in the cytoplasm by complex biosynthetic pathways, after which they are transported to the apoplastic space, where they are polymerized via free radical coupling reactions into polymeric lignin. However, the lignin deposition process and the factors controlling it are unclear. In this study, the biochemical and developmental dependencies of lignification were investigated using a click-compatible monolignol analog, 3-O-propargylcaffeyl alcohol (3-OPC), which can incorporate into both in vitro polymerized lignin and Arabidopsis thaliana tissues. Fluorescence labeling of 3-OPC using click chemistry followed by confocal fluorescence microscopy enabled the detection and imaging of 3-OPC incorporation patterns. These patterns were consistent with endogenous lignification observed in different developmental stages of Arabidopsis stems. However, the concentration of supplied monolignols influenced where lignification occurred at the subcellular level, with low concentrations being deposited in cell corners and middle lamellae and high concentrations also being deposited in secondary walls. Experimental inhibition of multiple lignification factors confirmed that 3-OPC incorporation proceeds via a free radical coupling mechanism involving peroxidases/laccases and reactive oxygen species (ROS). Finally, the presence of peroxide-producing enzymes determined which cell walls lignified: adding exogenous peroxide and peroxidase caused cells that do not naturally lignify in Arabidopsis stems to lignify. In conclusion, 3-OPC accurately mimics natural lignification patterns in different developmental stages of Arabidopsis stems and allows for the dissection of key biochemical and enzymatic factors controlling lignification.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2016

Extended-Charge-Transfer Excitons in Crystalline Supramolecular Photocatalytic Scaffolds

Coupling among chromophores in molecular assemblies is responsible for phenomena such as resonant energy transfer and intermolecular charge transfer. These processes are central to the fields of organic photovoltaics and photocatalysis, where it is necessary to funnel energy or charge to specific regions within the system. As such, a fundamental understanding of these transport processes is essential for developing new materials for photovoltaic and photocatalytic applications. Recently, photocatalytic systems based on photosensitizing perylene monomimide (PMI) chromophore amphiphiles were found to show variation in hydrogen gas (H 2 ) production as a function of nanostructure crystallinity. The 2D crystalline systems form in aqueous electrolyte solution, which provides a high dielectric environment where the Coulomb potential between charges is mitigated. This results in relatively weakly bound excitons that are ideal for reducing protons. Furthermore, in order to understand how variations in crystalline structure affect H 2 generation, two representative PMI systems are investigated theoretically using a modified Holstein Hamiltonian. The Hamiltonian includes both molecular Frenkel excitations (FE) and charge-transfer excitations (CTE) coupled nonadiabatically to local intramolecular vibrations. Signatures of FE/CTE mixing and the extent of electron/hole separation are identified in the optical absorption spectrum and are found to correlate strongly to the observed H 2 production rates. The absorption spectral signatures are found to sensitively depend on the relative phase between the electron and hole transfer integrals, as well as the diabatic energy difference between the Frenkel and CT exciton bands. Our analysis provides design rules for artificial photosynthetic systems based on organic chromophore arrays.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2016

Controlling Proton Delivery with Catalyst Structural Dynamics

The fastest synthetic molecular catalysts for production and oxidation of H2 emulate components of the active site of natural hydrogenases. The role of controlled structural dynamics is recognized as a critical component in the catalytic performance of many enzymes, including hydrogenases, but is largely neglected in the design of synthetic molecular cata-lysts. In this work, the impact of controlling structural dynamics on the rate of production of H2 was studied for a series of [Ni(PPh2NC6H4-R2)2]2+ catalysts including R = n-hexyl, n-decyl, n-tetradecyl, n-octadecyl, phenyl, or cyclohexyl. A strong correlation was observed between the ligand structural dynamics and the rates of electrocatalytic hydrogen production in acetonitrile, acetonitrile-water, and protic ionic liquid-water mixtures. Specifically, the turnover frequencies correlate inversely with the rates of ring inversion of the amine-containing ligand, as this dynamic process dictates the positioning of the proton relay in the second coordination sphere and therefore governs protonation at either catalytically productive or non-productive sites. This study demonstrates that the dynamic processes involved in proton delivery can be controlled through modifications of the outer coordination sphere of the catalyst, similar to the role of the protein architecture in many enzymes. The present work provides new mechanistic insight into the large rate enhancements observed in aqueous protic ionic liquid media for the [Ni(PPh2NR2)]2+ family of catalysts. The incorporation of controlled structural dynamics as a design parameter to modulate proton delivery in molecular catalysts has enabled H2 production rates that are up to three orders of magnitude faster than the [Ni(PPh2NPh2)]2+complex. The observed turnover frequencies are up to 106 s-1 in acetonitrile-water, and over 107 s-1 in protic ionic liquid-water mixtures, with a minimal increase in overpotential. This material is based upon work supported as part of the Center for Molecular Electrocatalysis, an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, and was performed in part using the Molecular Science Computing Facility (MSCF) in the William R. Wiley Environmental Molecular Sciences Laboratory, a DOE national scientific user facility located at Pacific Northwest National Laboratory (PNNL). PNNL is operated by Battelle for DOE.

electrocatalysis, structural dynamics↗

DOE OSTI2017

Interplay between Terminal and Bridging Diiron Hydrides in Neutral and Oxidized States

This study describes the structural, spectroscopic, and electrochemical properties of electronically unsymmetrical diiron hydrides. The terminal hydride Cp*Fe(pdt)Fe(dppe)(CO)H ([1(t-H)]0, Cp*– = Me5C5 –, pdt2– = CH2(CH2S–)2, dppe = Ph2PC2H4PPh2) was prepared by hydride reduction of [Cp*Fe(pdt)Fe(dppe)(CO)(NCMe)]+. As established by X-ray crystallography, [1(t- H)]0 features a terminal hydride ligand. Unlike previous examples of terminal diiron hydrides, [1(t-H)]0 does not isomerize to the bridging hydride [1(µ-H)]0. Oxidation of [1(t-H)]0 gives [1(t- H)]+, which was also characterized crystallographically as its BF4 – salt. Density functional theory (DFT) calculations indicate that [1(t-H)]+ is best described as containing an Cp*Fe(III) center. In solution, [1(t-H)]+ isomerizes to [1(µ-H)]+ as anticipated by DFT. Reduction of [1(µ- H)]+ by Cp2Co affords the diferrous bridging hydride [1(µ-H)]0. Electrochemical measurements and DFT calculations indicate that the couples [1(t-H)]+/0 and [1(µ-H)]+/0 differ by 210 mV. Qualitative measurements indicate that [1(t-H)]0 and [1(µ-H)]0 are close in free energy. Protonation of [1(t-H)]0 in MeCN solution affords H2 even with weak acids via hydride transfer.

Yu, Xin↗

DOE OSTI2018

Electron Transfer to Nitrogenase in Different Genomic and Metabolic Backgrounds

Nitrogenase catalyzes the reduction of dinitrogen (N 2 ) using low-potential electrons from ferredoxin (Fd) or flavodoxin (Fld) through an ATP-dependent process. Since its emergence in an anaerobic chemoautotroph, this oxygen (O 2 )-sensitive enzyme complex has evolved to operate in a variety of genomic and metabolic backgrounds, including those of aerobes, anaerobes, chemotrophs, and phototrophs. However, whether pathways of electron delivery to nitrogenase are influenced by these different metabolic backgrounds is not well understood. Here, we report the distribution of homologs of Fds, Flds, and Fd-/Fld-reducing enzymes in 359 genomes of putative N 2 fixers (diazotrophs). Six distinct lineages of nitrogenase were identified, and their distributions largely corresponded to differences in the host cells' ability to integrate O 2 or light into energy metabolism. The predicted pathways of electron transfer to nitrogenase in aerobes, facultative anaerobes, and phototrophs varied from those in anaerobes at the levels of Fds/Flds used to reduce nitrogenase, the enzymes that generate reduced Fds/Flds, and the putative substrates of these enzymes. Proteins that putatively reduce Fd with hydrogen or pyruvate were enriched in anaerobes, while those that reduce Fd with NADH/NADPH were enriched in aerobes, facultative anaerobes, and anoxygenic phototrophs. Here, the energy metabolism of aerobic, facultatively anaerobic, and anoxygenic phototrophic diazotrophs often yields reduced NADH/NADPH that is not sufficiently reduced to drive N 2 reduction. At least two mechanisms have been acquired by these taxa to overcome this limitation and to generate electrons with potentials capable of reducing Fd. These include the bifurcation of electrons or the coupling of Fd reduction to reverse ion translocation.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2018

DOE BES: Directed Assembly of Bio-inspired Supramolecular Materials for Energy Transport and Capture: Mesoscale Construction of Functional Materials in Hydrodynamic Flows. Final Project Report

The precise engineering of mesoscale structures based on molecular building blocks holds enormous promise to catalyze the development of new functional materials for advanced energy technologies. Bio-inspired and synthetic/bio-hybrid systems can play a key role in this effort due to their inherent programmable function and high degrees of sequence control. Despite recent progress, the ability to precisely control the supramolecular assembly of bio-hybrid molecules remains a central problem in the field. Our research aims to fundamentally understand the assembly mechanism of bio-hybrid materials, specifically focusing on sequence-controlled synthetic pi-conjugated oligopeptides. Moreover, we aim to develop new methods for the directed assembly of materials based on tailored fluid flows to guide the emergent structural, optical, and electronic properties of synthetic pi-conjugated peptides. Overall, our team has made substantial progress on this project through four major Thrusts: (1) Understanding the role of kinetics on the assembly process of synthetic π-conjugated peptides. (2) Probing the role of chemistry on the structural properties of these materials. (3) Real-time in situ investigation of the sol-gel transition and assembly of π-conjugated peptides. (4) Achieving large-area macroscopic alignment and assembly of these materials. In the following report, we explain major research progress in each of these research Thrusts.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2019

Dynamical Self-Assembly: Constrained phase separation and mesoscale dynamics in lipid membranes (Final Report)

This final technical report summarizes scientific and technical achievements derived through the the award, DE-FG02-04ER46173 [2013-2016, 2016-2017, and 2017-2018(NCE)], for the grant proposal titled, “Dynamical Self-Assembly: Constrained phase separation and mesoscale dynamics in lipid membranes.” The effort was organized into three major scientific aims: (1) characterize the mechanism of inter-layer coupling of laterally phase-separated domains in stacked lipid bilayers; (2) characterize curvature-dependent dynamic reorganization in membrane milieu; and (3) characterize real-time effects of mechanochemical perturbations on lateral phase separation membrane bilayers. The cumulative weight of the activities, including experimental research and collaborative modeling, disseminated through the publications listed above and presentations, has addressed two over-arching objectives: (a) to abstract a physical science based understanding of fundamental rules that determine dynamic self-assembly processes in lipid bilayer membranes - a versatile class of biologically inspired interfacial complex fluids and (b) to translate these physical principles into quantitative design rules for the development of new classes of membrane-based complex materials that exhibit complex, co-operative, and adaptive behavior.

36 MATERIALS SCIENCE↗

DOE OSTI2021

Maturation of Rhodobacter capsulatus Multicopper Oxidase CutO Depends on the CopA Copper Efflux Pathway and Requires the cutF Product (Final Report)

The goal is to achieve a comprehensive understanding of the nature, mechanism of function and biogenesis of c-type cytochromes and their oligomeric complexes. These cytochrome are ubiquitous heme proteins that are required for cellular bioenergetics, including photosynthesis (Ps) and respiration (Res), which are central to chemical energy (ATP) production by oxidative- and photophosphorylation. We contribute to the molecular characterization of individual and membrane-integral cytochromes as parts of Ps and Res electron transport (ET) pathways, using the facultative phototrophic bacteria of as excellent models for multidisciplinary investigations. Supported by this grant we discovered novel membrane-bound cytochromes and cyts c complexes that are important for Ps- and Res-ET. We worked out their biogenesis including maturation (covalent heme addition) and assembly of mature subunits into complexes. These involve fundamental process found virtually in all organisms and are fundamental processes with major significance to the “Photosynthetic Systems” program of DOE-BES, because the cytochromes are integral components of natural energy production systems. Elucidation of basic principles, and the knowledge gained by their study greatly impact on our ability to design, engineer, and create new natural, bio-hybrid, bio-inspired and artificial systems to better capture light, transduce and store its energy with improved efficacy and are of broad significance.

14 SOLAR ENERGY↗

NASA NTRS2004

Adaptive Bio-Inspired Wireless Network Routing for Planetary Surface Exploration

Wireless mobile networks suffer connectivity loss when used in a terrain that has hills, and valleys when line of sight is interrupted or range is exceeded. To resolve this problem and achieve acceptable network performance, we have designed an adaptive, configurable, hybrid system to automatically route network packets along the best path between multiple geographically dispersed modules. This is very useful in planetary surface exploration, especially for ad-hoc mobile networks, where computational devices take an active part in creating a network infrastructure, and can actually be used to route data dynamically and even store data for later transmission between networks. Using inspiration from biological systems, this research proposes to use ant trail algorithms with multi-layered information maps (topographic maps, RF coverage maps) to determine the best route through ad-hoc network at real time. The determination of best route is a complex one, and requires research into the appropriate metrics, best method to identify the best path, optimizing traffic capacity, network performance, reliability, processing capabilities and cost. Real ants are capable of finding the shortest path from their nest to a food source without visual sensing through the use of pheromones. They are also able to adapt to changes in the environment using subtle clues. To use ant trail algorithms, we need to define the probability function. The artificial ant is, in this case, a software agent that moves from node to node on a network graph. The function to calculate the fitness (evaluate the better path) includes: length of the network edge, the coverage index, topology graph index, and pheromone trail left behind by other ant agents. Each agent modifies the environment in two different ways: 1) Local trail updating: As the ant moves between nodes it updates the amount of pheromone on the edge; and 2) Global trail updating: When all ants have completed a tour the ant that found the shortest route updates the edges in its path.

Alena, Richard I.↗